Protein expression in Baculovirus system
Next to its microbial expression capabilities, Protéus now provides a service for evaluating the expression of your proteins in Baculovirus system.
Baculovirus Expression Vector System (BEVS) is utilized for producing recombinant proteins in insect cells. This superior eukaryotic system enables to produce proteins with very similar properties than the mammalian systems (folding, glycosylations...).
Another major advantage of baculovirus is the possibilities to produce monoclonal antibodies and complex eukaryotic proteins for research purposes.
The system also provides high expression yields and is considerably more cost-effective and rapid than mammalian cell expression. Finally, Baculovirus is non-pathogenic and has been FDA-approved for cGMP manufacturing. Vaccines produced in the baculovirus system have already been approved on market.
Baculovirus expression services
Each protein requires specific conditions for expression and purification, and these conditions are little predictable.
The expression feasibility step allows to rapidly identify suitable conditions for expressing your protein and evaluate production and purification performances.
1. Construction of a recombinant virus
Your gene is cloned in appropriate Proteus' vector to allow the subsequent expression profiling in insect cells. A tag can be added in the construct to facilitate the detection and the purification of the protein after production, as well as a proteolytic cleavage site. The vector is then co-transfected in Sf9 cells with baculoviral DNA and clones of recombinant viruses are selected. Each clone is screened for the expression of your protein using western blot.
Deliverables:
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Results of Western blotting for each selected clone
Results of Quality Controls (Sequencing...)
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2. Preparation of high titer viral stocks & mini-test of optimal expression
This step includes viral amplification of the positive viral clones identified during step 1on sf9 cells. The viral titer is quantified (pfu/mL). This viral stock will then be available for future protein productions. Small-scale expression will be performed in two different cell-lines. Cells and supernatant will be separated. Both fractions are analysed by western blotting to allow the choice of the best cell-line.
Deliverables:
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Titer of the final viral stock
Results of Western blotting for each cell-line and each fraction
Suggestion of the cell-line to be used
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3. Small-scale validation
A small-scale expression (100mL) is then performed in the two condition. Provided that a tag has been added to the construct, the protein is purified by affinity chromatography. The global yield after production and purification is then evaluated by western blotting.
The remaining amount of purified protein can then be transferred to you for validation.
Optionally, Protéus can also provide MALDI analysis of your protein.
This step allows to determine the culture volume that will be necessary to produce the amount of protein you need.
4. Protein production
With the viral stock obtained and the expression yield measured in the selected cell-line, Protéus will produce the protein in the appropriate culture volume to get the required quantity of the protein for the final application. Protéus provides culture capabilities from 100 mL to 10 L. The production is controled by Western blot.
Deliverables:
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Results of Quality controls
Pellets and/or supernatants obtained with this culture volume
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